Incipient spontaneous granulosa cell tumour

in the gerbil, Meriones unguiculatus

M A Guzmán-Silva and M Costa-Neves

Department of Pathology, Federal Fluminense University, Niteroi, RJ, Brazil

Summary
In 167 cases of granulosa cell tumour (GCT) in the gerbil, Meriones unguiculatus, 42.5%
(71 cases) were in the ovaries, with no evidence of macroscopic lesion. In this paper, we
describe the common findings in the incipient – microscopic – type of this spontaneous
ovarian tumour occurring in gerbils younger than two years of age.

Keywords Gerbil; granulosa cell tumour; ovary

The granulosa cell tumour (GCT), one of Materials and methods

the tumours of the theca-granulosa cells, is
Seventy-one cases of incipient GCT in
one of a group of neoplasms derived from
gerbils were analysed, without any
the strome of the sexual cord (Crum 2000,
macroscopic alteration of the ovaries. The
Miranda et al. 2000). These tumours
animals were maintained in the Animal
originate from the ovarian mesenchyme
Facility of the Section of Experimental
and have clinical importance, once they
Pathology in the Department of Pathology
synthesize great amounts of oestrogens, or
at the Federal Fluminense University. All
they can have malignant effects (Crum 2000,
the animals received commercial food and
Miranda et al. 2000). In addition, it was
filtered water ad libitum. Animals were
observed that, in specific cells of ovary
monitored five days a week, and no signs
carcinomas, they can produce a great
of sickness, discomfort or distress were
quantity of steroid hormones, which
noticed. The gerbils were classified into
explains the virilizing effect of certain GCTs
virgins and reproducers and distributed in
(Crum 2000).
three age groups for the analysis. After
As the spontaneous GCT in the gerbil
complete necropsy, fragments of several
reproduces in some respects the human
organs were collected besides both ovaries,
ovarian neoplasm, its use as a model of the
fixed in 10% buffered formalin, and
disease allows the study of several aspects,
processed for embedding in paraffin, as
such as tumour growth and progression,
is usual in histopathology; 5 mm sections
development and metastasis pattern,
were stained with haematoxylin and eosin.
therapeutic procedures, and hormone and
Besides the microscopic characteristics
immune modulation.
of incipient GCT, the presence, frequency
The aim of this study was to define
and metastases location were analysed,
the biological characteristics – incidence,
defining whether the GCT was malign
macroscopy, histopathology and metastases
or not.
– of the incipient type of GCT in the gerbil.
The testosterone level was analysed in
22 cases of bilateral incipient GCT. The
Correspondence: Maria Angelica Guzman-Silva, Rua Pereira
Da Silva 137/1806, 24220-030 Icarai, Niteroi, RJ, Brazil.
blood was collected from the retro-orbital
Email: mangie@ajato.com.br sinus vein, with the animals properly
Accepted 21 April 2005 r Laboratory Animals Ltd. Laboratory Animals (2006) 40, 96–101
Incipient granulosa cell tumour in the gerbil
anaesthetized by ether inhalation before
being killed through cervical dislocation.
The serum was stored at 201C for
the androgen test by the solid phase
radioimmunoassay technique (Samanta &
Ali 1990). Serum obtained from normal
males and females was used as a control.
Results
Forty-six incipient tumours were bilateral,
representing 64.8% of the total incipient
GCTs. Unilateral incipient GCT occurred in
the right ovary (RO) in 17 cases (23.9%) and
in the left ovary (LO) in eight cases (11.3%).
The frequency by sexual status was 44 in
virgins (62%) and 27 in reproducers (38%).
The age group with the larger incidence of
incipient GCT was the one of 1–2 years, with
36 cases (50.7%). Virgins from 1–2 years
acccounted for 34 cases (47.9%) and
reproducers from 2–3 years for 17 cases
(23.9%) (Figure 1).
Microscopically, GCT was characterized
as unilateral incipient lesions in 25 cases
(35.2%) and bilateral incipient lesions in 46
cases (64.8%), giving a total of 117 tumours
(Figure 2). The tumours were mainly
composed of nests and cords of cubical
tumour cells, some were observed in the
cystic wall and others substituted the
germinative epithelium (Figure 3A). The
other findings were luteinic cells (RO 56;
LO 47) (Figure 3B), incipient cysts (RO 50;
LO 41), Call–Exner bodies (RO 16; LO 12)
40 Reproducer
35 Virgin
30
25
20
n
15
10
5
0 investigation of gerbils from the stock of our
1–2 years 2–3 years > 3 years
Age
Figure 1 Distribution by age and sexual status of
71 incipient granulosa cell tumour in gerbils
97
Precocious tumour lesion
Cysts
Call-Exner bodies Right ovary
Left ovary
Pseudofollicles
Luteinic cells
0 20 40 60 80 100 120
n
Figure 2 Microscopic characteristics of 117 incipient
granulosa cell tumours of gerbils distributed by
anatomic location
and pseudofollicles (RO 4; LO 3) (Figure 3C).
The frequency of luteinic cells varied from
low to high, and a high number was present
in most of the cases. Some cysts were
already well developed. Mitosis was not
observed.
Local invasion was detected in the ovarian
hilus in 54 cases (RO 49; LO 34) (Figure 3D),
periovarian fat in 15 cases (RO 15; LO 11),
fimbriae in 17 cases (RO 13; LO 11) (Figure
3E) and large ligaments in three cases
(Figure 4).
Metastases occurred in the abdomen, the
omentum being the most affected organ (30
cases) (Figure 3F), followed by the mesentery
(three cases). Metastases were not found in
the thorax.
Serum testosterone was determined in 14
virgins and eight reproducers with bilateral
incipient GCT, and the values varied
between 0 and 4.80 ng/mL. In the virgins, the
mean value was 1.30 ng/mL and, in the
reproducers, it was 0.40 ng/mL. When the
values were analysed by age, it was found
that the average for the group of 1–2 years
was 1.30 ng/mL, 0.35 ng/ml for the group
of 2–3 years and 0.70 in the older group.
No statistical difference was detected
(Mann–Whitney test).
Discussion
Our data on this neoplasm were obtained
through systematic necropsy and exhaustive
colony.
Spontaneous ovarian and other tumours
can appear in gerbils from two years of age
(Benitz & Kramer Jr 1965, Rowe et al. 1974,
Laboratory Animals (2006) 40
98 M A Guzmán-Silva & M Costa-Neves

Figure 3 Incipient granulosa cell tumour of the gerbil (haematoxylin and eosin stain). (A) Tumour nest of
cuboidal cells substituting the ovarian germinative epithelium. (B) Tumour nest and cords of luteinic cells.
(C) Call–Exner body (arrow) and pseudofollicles. (D) Tumour invasion of the ovarian helium. (E) Tumour
invasion of the de fimbriae. (F) Incipient granulosa cell tumour metastases in the omentum

Vincent & Ash 1978, Meckley & Zwicker
1979, Vincent et al. 1979, Guzmán-Silva &
Rossi 1990, Guzmán-Silva et al. 1995a,
Matsuoka & Suzuki 1995). In this sampling,
we verified the occurrence of GCT in gerbils
Laboratory Animals (2006) 40
aged less than two years. Most of the
incipient tumours were bilateral, represen-
ting 71 (64.8%) of the total, 44 of them in
virgins (62%) and 27 in reproducers (38%).
Incipient GCT can appear precociously in
Incipient granulosa cell tumour in the gerbil
Indeterminate
Large ligament
Left ovary
Right ovary
Fimbriae
Periovarian fat
Ovarian hilus
0 10 20 30 40
n
Figure 4 Distribution by anatomic location of local
invasion of 71 cases of incipient granulosa cell
tumour in gerbils
gerbils of 1–2 years old. As the animal grows,
these cystic and solid tumours can develop
(dates not presented). The aetiology of
GCT is still unclear. Epidemiological data
indicate an increased incidence of ovarian
epithelial cancer, with early menarche, late
menopause and nulliparity. GCT is both
regulated by and can synthesize hormones; it
therefore seems logical that the molecular
changes which occur in a granulosa cell and
lead to its neoplastic transformation involve
these endocrine pathways (Fuller & Chu
2004). The higher incidence of GCT in
virgins could be explained by pregnancy
protecting against GCT development, in
rodents as well as in humans, since it causes
a break in the cyclical action of FSH.
Microscopically, gerbil GCT is composed
of cubical cells in diffuse arrangement, in
nests and cords, cysts, or with a follicular
pattern, as well as mitosis (Benitz & Kramer
Jr 1965, Ringler et al. 1972, Meckley &
Zwicker 1979). The same features are
observed in human GCT (Kurman et al.
1979, Chadha et al. 1990, Haba et al. 1993).
Haemorrhagic cysts of several sizes have
been described in the GCT of gerbils
(Meckley & Zwicker 1979) and mice
(Beamer et al. 1985). A prevalence of
vacuolated luteinic cells has been described
in gerbils (Benitz & Kramer Jr 1965, Ringler
et al. 1972, Rowe et al. 1974). Call–Exner
bodies have only been described in human
GCT (Kurman et al. 1979, Chadha et al.
1990), but we have identified them in gerbil
GCT. The microscopic aspects of the
99
incipient tumours were characterized by
tumour cells in nests and cords; some of
which were observed in the cystic wall, and
with others substituting the germinative
epithelium. Luteinic cells and incipient
cysts were observed as well as Call–Exner
bodies. Mitosis was not observed, probably
due to a low proliferative rate during the
50
precocious phase of the tumour development.
The gerbil ovary presented histo-
pathological alterations of GCT, mainly
related to luteinization of the tumour cells,
even if no ovarian alteration was observed
macroscopically. Therefore, the study of
precocious lesions of this tumour is very
important, as they can result in significant
hormonal alterations. Long-term studies
in this species should be attentive to the
interference of this pathology.
Only one reference describes the invasion
of GCT in the uterine serosa and myo-
metrium of gerbils (Rowe et al. 1974).
Local invasion of incipient GCT was most
frequent in the ovarian hilus (54 cases),
followed by periovarian fat and fimbriae.
The largest number of metastases
occurred in the abdominal omentum (30
cases). Our data agree with that described
in mice (Beamer et al. 1985). Only one
reference tells about the absence of
metastases in GCT of gerbils (Meckley &
Zwicker 1979). Metastases of GCT have
already been observed in abdominal
organs such as lymph nodes, kidneys,
pancreas, liver and diaphragm (Beamer
et al. 1985).
Considering the local invasion and
metastases, most of the incipient GCT in
gerbils (84.5%) was classified as malignant
tumours. Only four bilateral and seven
unilateral GCT did not show malignant
behaviour by microscopy.
In the incipient GCT, the serological
level of testosterone varied between 0 and
4.80 ng/mL in 22 cases, and the average
was 1.80 ng/mL. By sexual status and age,
the average was higher in virgins of less
than two years old (1.30 ng/mL). In normal
gerbils, using the same methodology for
the detection of serological testosterone, we
found an average of 0.13 ng/mL in females
and 0.82 ng/mL in males (Guzmán-Silva
Laboratory Animals (2006) 40
100
et al. 1995b). Since the mean value found
in females carrying incipient GCT was
higher than that in normal males, it seems
that a high level of testosterone could be
related to GCT development in gerbils.
Androgens acting synergistically to FSH
interfere in the lipoprotein metabolism in
ovarian granulosa cells (Schreiber et al.
1984). The same synergism could play a
role in the regulation of proliferation in
those cells, which would have some contri-
bution to tumorigenesis.
In human GCT, the immunoreactivity of
oestradiol and progesterone has been
detected more constantly and the results
related to testosterone have already been
recognized (Kurman et al. 1979, Chadha
et al. 1990, Mulvany & Riley 1997). In
humans, testosterone production by GCT
cells is associated with several degrees of
virilization (Kurman et al. 1979, Chadha
et al. 1990). In the gerbil GCT secreting
androgens can apparently present a virilizing
behaviour.
The histopathological findings of gerbil
GCT resemble the ones observed in the GCT
of rodents and humans. The initial spreading
of the tumour begins in the peritoneal
cavity, and metastases probably occur in
the abdominal organs due to lymphatic and
haematogenous spread. Incipient GCT in
gerbils occurs in animals of less than two
years of age, mainly in virgins. This
precocious tumour is similar to that of the
human ovarian neoplasm, and can be used as
an animal model of the disease. It would
therefore facilitate future studies of genetic,
hormonal and immune modulation, and the
action of antineoplasmic drugs during the
whole period of the ovarian tumour
development.
Acknowledgement The authors are grateful to the
Conselho Nacional de Pesquisa e Desenvolvimento
(CNPq) for the support during this research.
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